ABSTRACT Now it is unambiguous that antimicrobial resistance increases the morbidity, mortality and costs of treating infectious diseases. There are two critical elements needed that can reduce the negative impact of multidrug resistant organisms (MDROs). One is rapid detection of colonized patients so that the spread of MDROs in healthcare facilities can be curtailed. The other is to rapidly detect them when a MDRO is causing infection so that patients are placed on appropriate therapy early in their disease. Current examples of this important group of microbes, due to their either frequency in causing infection, the lack of antimicrobial agents effective against them, and/or ability rapidly acquire broad range of resistance include methicillin-resistant S. aureus (MRSA), vancomycin-resistant enterococci (VRE), carbapenemase-producing K. pneumonia (KPCs), and A. baumannii. The major source of resistance to &#946;-lactam antibiotics occurs through production of &#946;-lactamases. One of these enzyme groups now readily spreading, KPCs, cause infections that are exceptionally difficult to treat and have high mortality rates. This is becoming more complicated due to their appearance in many MDROs. Hence, a fast and reliable diagnostic tool remains urgently needed. Traditional clinical microbiological approaches are excessively time consuming, difficult in interpretation and suffer from suboptimal sensitivity/specificity. The objective of this project is to develop, evaluate, and validate a broad-spectrum assay for fast and reliable molecular identification of MDROs (including but not limited to KPCs, A. baumannii, and VRE) in both surveillance swabs that detect colonization plus clinical specimens that diagnose disease in conjunction with exploring and evaluating of two novel, highly multiplex and specific approaches, sloppy molecular beacons and RAIN probes. The concept for the proposed application is a) to utilize surveillance swabs and a real-time PCR for our proved successful approach of in vitro KPC identification;b) employ sloppy beacons and RAIN probes incorporated with real-time PCR amplification for highly specific DNA detection of multiple pathogenic microbes;and c) integrate real-time PCR based assays with well established Cepheid technology. To accomplish the objectives of this application, we will pursue three specific aims: 1) develop a rapid and reliable KPC detection assay for direct deployment in clinical practice;2) develop and explore two innovative novel approaches (sloppy beacons and RAIN probes) for rapid and highly specific detection of A. baumannii in both surveillance and clinical infection specimens;3) Incorporate the demonstrated success of the methodology for development of a broad range single cartridge test for KPC, VRE, and A. baumannii detection within 1 hour in surveillance and clinical samples integrated with Cepheid[unreadable]s easy to use GeneXpert platform. When successfully developed we will expand our assay to other important pathogens such as MDR P. aeruginosa and Enterobacteriaceae producing extended spectrum &#946;-lactamases. Our long term goal is development of a point-of-care broad-spectrum diagnostic platform for rapid and accurate identification of pathogenic MDROs in surveillance and clinical samples.